Methodologies have evolved during the last twenty years to genetically engineer plants. In general, they are based on either direct DNA introduction into plant cells or indirect transfer mediated by Agrobacterium tumefaciens. Methods involving direct transfer include particle bombardment of cultured plant tissues and DNA introduction into naked plant cells i.e., protoplasts, using polyethylene glycol or electroporation. See, e.g., Sawahel & Cove, Biotech, Adv. 10:394–412 (1992); Christou, Cur. Opinion Biotech. 4:135–141 (1993); Gelvin, Cur. Opinion Biotech. 9:227–232 (1998) and Birch, Annu. Rev. Plant Physiol. Plant Mol. Biol, 48:297–326 (1997). Most methods are variety-specific because they are based on use of in vitro grown regenerable plant systems which in turn are variety-specific. Except for a few economically important crops such as potato, tomato and canola, transformation methods available currently work with only a handful of varieties.
The traditional backcross method of breeding has provided a mechanism for the transfer of a trait from one line (the donor) to another line (the recurrent parent). See, e.g., Harlan and Pope, J. Heredity, 13:319–322 (1922). It has been particularly useful for corn, soybean and cotton. Successful backcross breeding requires: a previously derived recurrent parent; maintenance of the trait of interest during selection; and sufficient backcrosses to reconstitute the genome of the recurrent parent. Allard, Principles of Plant Breeding, Wiley and Sons (1960). During the backcross program, the hybrid population becomes increasingly homozygous for genes of the recurrent parent at a rate described by the formula:Proportion of homozygosity=1–0.5m where m is the number of backcross generations. Using this formula, one can calculate that more than 98% of the hybrid genome will be homozygous for genes of the recurrent parent after six generations. The formula, however, only describes regions of the genome that are unlinked with respect to the genes being introgressed. The rate at which linked regions approach homozygosity is dependent upon the chromosome recombination frequency. In one of the most detailed studies assessing the effectiveness of traditional backcross breeding, eight Tm-2-converted isogenic lines of tomato were examined at nine flanking restriction fragment length polymorphism (RFLP) loci. See, Young and Tanksley, Theor. Appl. Genet. 77:353–359 (1989). The minimum donor chromosome fragment found after 10 generations of backcrossing and maintained without reduction in size for an additional nine generations was 4 cM the maximum size found even after 11 generations was 51 cM (i.e., more than half of the corresponding chromosome). In marker-assisted selection based on simple sequence repeats (SSR) or RFLP, the reconstruction could be done faster and cleaner, but it would require screening of sizable populations of progeny using relatively expensive methods and would be complicated by the random insertion of transgenes in independent primary transformants.
Plainly, backcrossing is not a trivial task because for most crop plants, hundreds of lines, hybrids or varieties are needed simultaneously. In 1998 for example, the U.S. soybean seed market consisted of over 500 varieties, and the U.S. maize seed market included over 600 hybrids. Another major disadvantage of the backcrossing method is that it is very time-consuming. Line conversion through recurrent backcrossing normally requires 3 to 5 subsequent backcrosses, thus adding at least two and sometimes up to four years to variety development time.
There are many disadvantages associated with current transformation methods. Line conversion, for example, is a process whereby heterologous DNA is transferred from one plant species or variety to another using various forms of sexual (i.e., pollination) or somatic (i.e., cellular) hybridization. Because current methods are species- and variety-specific, they can be commercially used only in combination with line conversion technologies that allow for transgene transfer from a primary transformant into multiple varieties of interest. In addition, they result in random transgene insertion into the host genome. Therefore, extensive screening of numerous independent transformation events are required in order to identify the events that are stable, inheritable and allow for proper transgene expression. Subsequent transgene insertions cannot be addressed to the same site, thus complicating breeding. Linkage drag (i.e., co-inheritance of unwanted traits) and a limited ability to handle multiple independent transgene traits present even further difficulties.